Dipeptide production method, L-amino acid amide hydrolase used therein, and production method of L-amino acid amide hydrolase

ABSTRACT

A process for industrially advantageously producing a dipeptide via a convenient pathway starting with less expensive and easily available materials is provided. A dipeptide is produced from an L-amino acid amide and an L-amino acid by using a culture of a microbe capable of synthesizing the dipeptide from the L-amino acid amide and the L-amino acid, microbial cells separated from the culture or a treated microbial cell product from the microbe. An L-amino acid amide hydrolase is obtained from a microbe belonging to the genus  erwinia,  genus  Rhodococcus,  genus  Chryseobacterium,  genus  Micrococcus,  genus  Cryptococcus,  genus  Trichosporion,  genus  Rhodosporidium,  genus  Sporobolomyces,  genus  Tremela,  genus  Torulaspora,  genus  Sterigmatomyces  or genus  Rhodotorula.  The hydrolase catalyzes a reaction that produces a dipeptide from an L-amino acid amide and an L-amino acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. Ser. No. 10/481,087,filed on Dec. 17, 2003, which is a National Stage (371) ofPCT/JP02/07633 filed on Jul. 26, 2002, which claims priority to JP2001-226568, filed on Jul. 26, 2001, and to JP 2001-310547, filed onOct. 5, 2001.

TECHNICAL FIELD

The present invention relates to a method for producing dipeptideseasily and inexpensively without going through a complex synthesismethod, and more particularly, to a method for producing dipeptides froman L-amino acid amide and an L-amino acid, to an L-amino acid amidehydrolase used in the method for producing dipeptides, and to aproduction method of the same.

BACKGROUND ART

Dieptides are used in the field of pharmaceuticals and functional foodsand various other fields. For example, L-alanyl-L-glutamine is useful asa component of serum-free media, and since it has higher stability andwater-solubility than L-glutamine, it is used for fluid infusion.

Although chemical synthesis methods are conventionally known in the artas methods for producing dipeptides, those production methods are notnecessarily easy. Known examples of such methods include a method thatuses N-benzyloxycarbonylalanine (hereinafter, “Z-alanine”) and protectedL-glutamine (see Bull. Chem. Soc. Jpn., 34, 739 (1961), Bull. Chem. Soc.Jpn., 35, 1966 (1962)), a method that uses Z-alanine and protectedL-glutamic acid-γ-methyl ester (see Bull. Chem. Soc. Jpn., 37, 200(1964)), a method that uses a Z-alanine ester and unprotected glutamicacid (see Japanese Patent Application Laid-open Publication No.H1-96194), and a method that uses a 2-substituted-propionyl halide as araw material to synthesize a dipeptide using anN-(2-substituted)-propionyl glutamine derivative as an intermediate (seeJapanese Patent Application Laid-open Publication No. H6-234715).

However, since all of these methods require the introduction andelimination of protecting groups or the synthesis of an intermediate,they are not considered to be adequately satisfactory in terms of theirindustrial advantages.

In addition, known examples of methods for producing dipeptides usingmicrobial enzyme system include a method that uses Z-aspartic acid andmethyl ester of phenylalanine (see Japanese Patent Application Laid-openPublication No. S53-92729), and a method that uses aspartic acid amideand methyl ester of phenylalanine (see Japanese Patent ApplicationLaid-open Publication No. H10-136992). Other known examples of methodsfor producing dipeptides by an enzymatic process are described in EPA0278787 and WO 90/01555.

However, in all of these microbial enzyme systems, since it is necessaryto use an amino acid having a protecting group for the startingsubstance, there is a need to develop a method for producing dipeptidesthat uses raw materials that are available comparatively inexpensivelyand easily, is industrially advantageous and employs a simple productionpathway.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method forproducing dipeptides that uses comparatively inexpensive and easilyavailable starting materials, is industrially advantageous and employs asimple production pathway.

As a result of conducting extensive research in consideration of theaforementioned object, the inventors of the present invention found thatcertain microbes have the ability to form dipeptides from comparativelyinexpensive and easily available L-amino acid amides and L-amino acids,thereby having completed the present invention.

Namely, the present invention is as described below.

[1] A dipeptide production method comprising: producing a dipeptide froman L-amino acid amide and an L-amino acid using an enzyme orenzyme-containing substance having an L-amino acid amide hydrolaseactivity.

[2] The dipeptide production method according to [1] described above,wherein the enzyme or enzyme-containing substance is one or two or moretypes selected from the group consisting of a culture of microbes havingan L-amino acid amide hydrolase activity, microbial cells separated fromthe culture, and a treated microbial cell product from the microbes.

[3] The dipeptide production method according to [2] described above,wherein the microbe belongs to the genus Bacillus, genusCorynebacterium, genus Erwinia, genus Rhodococcus, genusChryseobacterium, genus Micrococcus, genus Pseudomonas, genusCryptococcus, genus Trichosporon, genus Rhodosporidium, genusSporobolomyces, genus Tremela, genus Torulaspora, genus Sterigmatomycesor genus Rhodotorula.

[4] The dipeptide production method according to [1] described above,wherein the enzyme is a protein (A) or (B):

(A) a protein having the amino acid sequence described in SEQ ID No.: 5of the Sequence Listing,

(B) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one or aplurality of amino acids in the amino acid sequence described in SEQ IDNo.: 5 of the Sequence Listing, and having an L-amino acid amidehydrolase activity that catalyzes a reaction that produces a dipeptidefrom the L-amino acid amide and the L-amino acid;

[5] The dipeptide production method according to [1] described above,wherein the enzyme is a protein encoded by a DNA of (C):

(C) a DNA that hybridizes under stringent conditions with apolynucleotide that consists of a base sequence complementary to thebase sequence of bases nos. 57 to 1295 described in SEQ ID No.: 4 of theSequence Listing, and encodes a protein having an L-amino acid amidehydrolase activity that catalyzes a reaction that produces a dipeptidefrom the L-amino acid amide and the L-amino acid.

[6] The dipeptide production method according to [2], wherein themicrobe is a microbe that has been transformed so as to be able toexpress the protein (A), (B) or (C):

(A) a protein having the amino acid sequence described in SEQ ID No.: 5of the Sequence Listing,

(B) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one or aplurality of amino acids in the amino acid sequence described in SEQ IDNo.: 5 of the Sequence Listing, and having an L-amino acid amidehydrolase activity that catalyzes a reaction that produces a dipeptidefrom the L-amino acid amide and the L-amino acid,

(C) a protein encoded by a DNA that hybridizes under stringentconditions with a polynucleotide that consists of a base sequencecomplementary to the base sequence of base nos. 57 to1295 described inSEQ ID No.: 4 of the Sequence Listing, and encodes protein having anL-amino acid amide hydrolase activity that catalyzes a reaction thatproduces a dipeptide from the L-amino acid amide and the L-amino acid.

[7] The dipeptide production method according to any one of [1] to [6],wherein the L-amino acid amide is one or two or more types selected fromthe group consisting of L-alanine amide, glycine amide and L-asparticacid-α-amide.

[8] The dipeptide production method according to any one of [1] to [7],wherein the L-amino acid is one or two or more types selected from thegroup consisting of L-glutamine, L-asparagine, glycine, L-alanine,L-valine, L-leucine, L-isoleucine, L-methionine, L-proline,L-phenylalanine, L-tryptophan, L-serine, L-threonine, L-tyrosine,L-lysine, L-arginine, L-histidine and L-glutamine.

[9] An L-amino acid amide hydrolase obtained from a microbe belonging tothe genus Erwinia, genus Rhodococcus, genus Chryseobacterium, genusMicrococcus, genus Cryptococcus, genus Trichosporon, genusRhodosporidium, genus Sporobolomyces, genus Tremela, genus Torulaspora,genus Sterigmatomyces or genus Rhodotorula, which catalyzes a reactionthat produces a dipeptide from an L-amino acid amide and an L-aminoacid.

[10] A production method of L-amino acid amide hydrolase, comprising:culturing a microbe belonging to the genus Erwinia, genus Rhodococcus,genus Chryseobacterium, genus Micrococcus, genus Cryptococcus, genusTrichosporon, genus Rhodosporidium, genus Sporobolomyces, genus Tremela,genus Torulaspora, genus Sterigmatomyces or genus Rhodotorula in amedium, and accumulating in the medium and/or cells an L-amino acidamide hydrolase that catalyzes a reaction that produces a dipeptide froman L-amino acid amide and an L-amino acid.

[11] A production method of L-amino acid amide hydrolase, comprising:culturing a microbe transformed so as to be able to express a protein(A), (B) or (C):

(A) a protein having the amino acid sequence described in SEQ ID No.: 5of the Sequence Listing,

(B) a protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one or aplurality of amino acids in the amino acid sequence described in SEQ IDNo.: 5 of the Sequence Listing, and having an L-amino acid amidehydrolase activity that catalyzes a reaction that produces a dipeptidefrom the L-amino acid amide and the L-amino acid; and

(C) a protein encoded by a DNA that hybridizes under stringentconditions with a polynucleotide that consists of a base sequencecomplementary to the base sequence of bases nos. 57 to 1295 described inSEQ ID No.: 4 of the Sequence Listing, and encodes a protein having anL-amino acid amide hydrolase activity that catalyzes a reaction thatproduces a dipeptide from the L-amino acid amide and the L-amino acid,and accumulating in the medium and/or cells an L-amino acid amidehydrolase that catalyzes a reaction that produces a dipeptide from anL-amino acid amide and an L-amino acid.

These and other objects, features and advantages of the presentinvention are specifically set forth in or will become apparent from thefollowing detailed descriptions of the invention when read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart of a dipeptide production method of the presentinvention;

FIG. 2 is a graph of the optimum pH curve of an L-amino acid amidehydrolase derived from Corynebacterium glutamicum ATCC 13286; and

FIG. 3 is a graph of the optimum temperature curve of an L-amino acidamide hydrolase derived from Corynebacterium glutamicum ATCC13286.

BEST MODE FOR CARRYING OUT THE INVENTION

The dipeptide production method of the present invention ischaracterized by using an enzyme or enzyme-containing substance havingthe ability to form a dipeptide from an L-amino acid amide and anL-amino acid, and more specifically, by using a culture of a microbehaving the ability to form a dipeptide from an L-amino acid amide and anL-amino acid, microbial cells separated from the culture, or a treatedmicrobial cell product from the microbe. The reaction in the dipeptideproduction method of the present invention is represented by thefollowing reaction formula. As exemplified in the following chemicalformula, the term “dipeptide” used in the present specification refersto a peptide polymer having one peptide bond.

(In the chemical formula, R¹ represents an amino acid side chain of anL-amino acid amide, while R² represents an amino acid side chain of anL-amino acid.)

Amino acid amides are compounds that can be acquired comparativelyinexpensively as commercially available products. The method of thepresent invention that uses an amino acid amide and an unprotected aminoacid as starting materials is able to inexpensively provide dipeptidesthat are useful as pharmaceutical materials and functional foods.

The following provides an explanation of the dipeptide production methodof the present invention with reference to the attached drawingspresented in the order of:

[I] Microbes having the ability to form dipeptides from L-amino acidamides and L-amino acids;

[II] Properties of L-amino acid amide hydrolase;

[III] Isolation of a DNA that encodes a protein having an L-amino acidamide hydrolase activity; and,

[IV] A dipeptide production method.

[I] Microbes Having the Ability to form Dipeptides From L-Amino AcidAmides and L-Amino Acids

Microbes having the ability to form dipeptides from L-amino acid amidesand L-amino acids can be used without restriction for the microbe usedin the present invention. Examples of microbes having the ability toform dipeptides from L-amino acid amides and L-amino acids includemicrobes belonging to the genus Bacillus, genus Corynebacterium, genusErwinia, genus Rhodococcus, genus Chryseobacterium, genus Micrococcus,genus Pseudomonas, genus Cryptococcus, genus Trichosporon, genusRhodosporidium, genus Sporobolomyces, genus Tremela, genus Torulaspora,genus Sterigmatomyces and genus Rhodotorula, and specific examples ofthese microbes are indicated below. Bacillus megateirum AJ3284 FERMBP-8090 Corynebacterium glutamicum ATCC13286 Erwinia carotovora AJ2719FERM BP-8089 Rhodococcus rhodochrous ATCC19149 Chryseobacteriummeningosepticum ATCC13253 Micrococcus luteus ATCC9341 Pseudomonassaccharophila ATCC15946 Cryptococcus albidus var. albidus IFO0378Trichosporon gracile ATCC24660 Rhodosporidium diobovatum ATCC22264Sporobolomyces salmonicolor IFO1038 Tremela foliacea IFO9297 Torulasporadelbrueckii IFO1083 Sterigmatomyces elviae IFO1843 Rhodotorula ingeniosaATCC22993

The depositary institutions of the aforementioned microbes are asindicated below.

The independent administrative corporation, International PatentOrganism Depositary, National Institute for Advanced Industrial Scienceand Technology, Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi,Ibaraki-ken, Japan

Institute for Fermentation, Osaka (IFO), 2-17-85 Jusanbon-cho,Yodogawa-ku, Osaka City, Japan

American Type Culture Collection, P.O. Box 1549, Manassas, Va., USA

Furthermore, Bacillus megaterium strain AJ3284 was deposited at theInternational Patent Organism Depositary of the independentadministrative corporation, National Institute for Advanced IndustrialScience and Technology on Jul. 13, 2001 and assigned the deposit numberof FERM P-18421. Control of this organism was subsequently transferredto deposition based on the Budapest Treaty at the International PatentOrganism Depositary of the independent administrative corporation,National Institute for Advanced Industrial Science and Technology onJun. 25, 2002 and was assigned the deposit number of FERM BP-8090. Inaddition, Erwinia carotovora strain AJ2719 was deposited at theInternational Patent Organism Depositary of the independentadministrative corporation, National Institute for Advanced IndustrialScience and Technology on Jul. 13, 2001 and assigned the deposit numberof FERM P-18420. Control of this organism was subsequently transferredto deposition based on the Budapest Treaty at the International PatentOrganism Depositary of the independent administrative corporation,National Institute for Advanced Industrial Science and Technology, ChuoDai-6, 1-1 Higashi 1-Chome , Tsukuba-shi, Ibaraki-ken, Japan on Jun. 25,2002 and was assigned the deposit number of FERM BP-8089.

Wild strains or variant strains may be used for these microbes, andrecombinant strains and so forth derived by cell fusion, geneticmanipulation or other genetic techniques may also be used.

It is recommended that these microbes be cultured and grown in asuitable medium in order to obtain microbial cells of these microbes.There is no particular restriction on the medium used for this purposeso far as it allows the microbes to grow. The medium may be an ordinarymedium that contains ordinary carbon sources, nitrogen sources,inorganic ions, and organic nutrient sources as necessary.

For example, any carbon source may be used so far as it can be utilizedby the microbes. Specific examples of the carbon source that can be usedinclude sugars such as glucose, fructose, maltose and amylose, alcoholssuch as sorbitol, ethanol and glycerol, organic acids such as fumaricacid, citric acid, acetic acid and propionic acid and their salts,hydrocarbons such as paraffin as well as mixtures thereof.

Examples of nitrogen sources that can be used include ammonium salts ofinorganic acids such as ammonium sulfate and ammonium chloride, ammoniumsalts of organic acids such as ammonium fumarate and ammonium citrate,nitrates such as sodium nitrate and potassium nitrate, organic nitrogencompounds such as peptones, yeast extract, meat extract and corn steepliquor as well as mixtures thereof.

In addition, ordinary nutrient sources used in media, such as inorganicsalts, trace metal salts and vitamins, can also be suitably mixed andused.

Microbial cells having a high level of activity to form dipeptides fromL-amino acid amides and L-amino acids may be obtained in some cases byfurther adding an L-amino acid amide to the medium.

There is no particular restriction on culturing conditions, andculturing may be carried out, for example, for about 12 to about 48hours while suitably controlling the pH and temperature to a pH range of5 to 8 and a temperature range of 20 to 40° C., respectively, underaerobic conditions.

[II] Properties of L-Amino Acid Amide Hydrolase

An explanation is provided of the properties of an L-amino acid amidehydrolase purified for use as an enzyme having activity to formdipeptides from L-amino acid amides and L-amino acids takingCorynebacterium glutamicum strain ATCC 13286 of the aforementionedmicrobes as an example.

The L-amino acid amide hydrolase has activity to form an L-amino acid byhydrolyzing and L-amino acid amide, and activity to form a dipeptide byusing an L-amino acid amide and an L-amino acid as substrates. Whentaking as an example the case of using L-alanine amide and L-glutamineas raw materials (substrates), the L-amino acid amide hydrolase at leasthas activity to form L-alanine by hydrolyzing L-alanine amide, andactivity to form L-alanyl-L-glutamine by using L-alanine amide andL-glutamine as substrates. In addition, in taking as an example the caseof using L-alanine amide and L-asparagine as raw materials, the L-aminoacid amide hydrolase at least has activity to form L-alanine byhydrolyzing L-alanine amide, and activity to form L-alanyl-L-asparagineby using L-alanine amide and L-asparagine as substrates.

With respect to the enzyme function, when taking as an example the caseof using L-alanine amide and L-glutamine or L-asparagine as rawmaterials, the L-amino acid amide hydrolase forms 1 molecule ofL-alanine and 1 molecule of ammonia by hydrolyzing 1 molecule ofL-alanine amide, forms 1 molecule of L-alanyl-L-glutamine and 1 moleculeof ammonia from 1 molecule of L-alanine amide and 1 molecule ofL-glutamine, and forms 1 molecule of L-alanyl-L-asparagine and 1molecule of ammonia from 1 molecule of L-alanine amide and 1 molecule ofL-asparagine.

The optimum pH is in the vicinity of 6.0 to 10.0, and the optimumtemperature is in the vicinity of 30° C. to 50° C. The molecular weightof the subunit is calculated to be 42,000 to 46,000 as determined bySDS-polyacrylamide gel electrophoresis.

[III] Isolation of a DNA Encoding a Protein Having an L-Amino Acid AmideHydrolase Activity

The enzyme or enzyme-containing substance that catalyzes a reaction thatproduces dipeptides from L-amino acid amides and L-amino acids used inthe present invention can be obtained by producing a transformant byisolating a DNA that encodes the enzyme using genetic engineeringtechniques from the aforementioned microbes having the enzyme.

For example, the following provides an explanation of a DNA encoding aprotein having an L-amino acid amide hydrolase activity isolated fromCorynebacterium glutamicum [III-1] along with a transformant in which ithas been inserted [III-2].

[III-1] DNA Isolation

First, the amino acid sequence is determined for the purified L-aminoacid amide hydrolase. The amino acid sequence can be determined usingthe Edman's method (see Edman, P., Acta Chem. Scand., 4, 227 (1950)). Inaddition, the amino acid sequence can also be determined using asequencer manufactured by Applied Biosystems. The amino acid sequence isdetermined for the N-terminal or about 10 to about 30 residues of thepeptide obtained by treatment with lysyl endopeptidase or the like forthe purified L-amino acid amide hydrolase, and the base sequence of aDNA that encodes the hydrolase can be deduced based on the determinedamino acid sequence. Universal codons are employed for deducing the basesequence of the DNA.

A DNA molecule of about 30 base pairs is synthesized based on thededuced base sequence. A method for synthesizing the DNA molecule isdisclosed in Tetrahedron Letters, 22, 1859 (1981). In addition, the DNAmolecule can also be synthesized using a synthesizer (manufactured byApplied Biosystems). DNA that encodes the L-amino acid amide hydrolasecan then be amplified from a chromosomal DNA by a PCR method using theDNA molecule as a primer. However, since the DNA that has been amplifiedusing the PCR method does not contain the full-length DNA that encodesthe L-amino acid amide hydrolase, the full-length DNA that encodes theL-amino acid amide hydrolase is isolated from a gene library using theDNA that is amplified by use of the PCR method as a probe.

Alternatively, in the case where a portion of the base sequence of thegene is known, the full-length DNA that encodes a peptide-forming enzymecan be isolated from a chromosomal gene library using a DNA having theknown sequence as a probe.

Moreover, in the case where the base sequence of the gene has homologywith a known sequence, the full-length DNA that encodes thepeptide-forming enzyme can be isolated from a chromosomal gene libraryusing a DNA having that known sequence as a probe.

A procedure for the PCR method is described in White, T. J. et al.,Trends Genet. 5, 185 (1989). A method for preparing a chromosomal DNA,as well as a method for isolating a target DNA molecule from a genelibrary using a DNA molecule as a probe, is described in MolecularCloning, 2nd edition, Cold Spring Harbor Press (1989).

A method for determining the base sequence of an isolated DNA thatencodes an L-amino acid amide hydrolase is described in A PracticalGuide to Molecular Cloning, John Wiley & Sons, Inc. (1985). In addition,the base sequence can also be determined using a DNA sequencer (AppliedBiosystems). A DNA that encodes the L-amino acid amide hydrolaseisolated from Corynebacterium glutamicum strain ATCC 13286 in thismanner is shown in SEQ ID No.: 4. The base sequence that consists ofbases nos. 57 to 1295 out of the base sequence of SEQ ID No.: 4 is a CDS(coding region) (in the present specification, the “base sequencedescribed in SEQ ID No.: 4” indicates a CDS portion unless otherwiseindicated specifically). Furthermore, although the amino acid amidehydrolase described in SEQ ID No.: 4 is inherently the result ofisolating a gene based on a purified enzyme using an alanine amidehydrolase activity as an indicator, it is referred to as amino acidamide hydrolase because its substrate specificity is not limited toalanine amide, but rather has an extremely broad spectrum.

A DNA that can be used in the present invention is not only the DNAspecified in SEQ ID No.: 4. With respect to the DNA of SEQ ID No.: 4isolated from Corynebacterium glutamicum strain ATCC 13286, even a DNAthat has been artificially mutated to a DNA that encodes the L-aminoacid amide hydrolase isolated from the chromosomal DNA ofCorynebacterium glutamicum strain ATCC 13286 is also a DNA of thepresent invention so far as it encodes an L-amino acid amide hydrolase.A frequently used method for artificially mutating a DNA is thesite-specific mutation introduction method described in Methods inEnzymol., 154 (1987).

In addition, a DNA having a base sequence that hybridizes with apolynucleotide having a base sequence complementary to the base sequenceof base nos. 57 to 1295 described in SEQ ID No.: 4 under stringentconditions, and encodes a protein having an L-amino acid amide hydrolaseactivity, can also used as a DNA in the present invention. The“stringent conditions” mentioned herein refer to conditions under whicha so-called specific hybrid is formed while non-specific hybrids are notformed. Although it is difficult to clearly quantify these conditions,examples of such conditions include conditions under which DNAs havinghigh degrees of homology, such as DNAs having a homology of 50% or more,preferably 80% or more, and more preferably 90% or more, hybridize witheach other while DNAs having low degrees of homology do not hybridizewith each other, and conditions under which DNAs hybridize at a saltconcentration equivalent to 60° C., 1×SSC and 0.1% SDS, preferably 60°C., 0.1×SCC and 0.1% SDS, and more preferably 65° C., 0.1×SSC and 0.1%SDS, which are the conditions for washing of ordinary Southernhybridization. The activity of L-amino acid amide hydrolase is aspreviously described. However, in the case of a base sequence thathybridizes under stringent conditions with a base sequence complementaryto the base sequence of bases nos. 57 to 1295 described in SEQ ID No.: 4of the Sequence Listing, it preferably retains about 10%, and preferablyabout 50% or more, of the enzyme activity of a protein having the aminoacid sequence described in SEQ ID No.: 5 of the Sequence Listing underconditions of 50° C. and pH 8.

Moreover, a protein substantially identical to the L-amino acid amidehydrolase encoded by the DNA described in SEQ ID No.: 4 of the SequenceListing can also be used in the present invention. Thus, a DNA thatencodes a “protein having an amino acid sequence containing asubstitution, deletion, insertion, addition or inversion of one or aplurality of amino acids in the amino acid sequence described in SEQ IDNo.: 5 of the Sequence Listing, and having the activity of an L-aminoacid amide hydrolase that forms a dipeptide from an L-amino acid amideand an L-amino acid” can also be used in the present invention. Here,the term “a plurality of” refers to a range that does not significantlyimpair the three-dimensional structure of the protein of the amino acidresidues or the activity of L-amino acid amide hydrolase, and morespecifically is a value of 2 to 50, preferably a value of 2 to 30, andmore preferably a value of 2 to 10. In addition, the activity of theL-amino acid amide hydrolase is as previously explained. However, in thecase of an amino acid sequence containing a substitution, deletion,insertion, addition or inversion of one or a plurality of amino acidresidues in the amino acid sequence described in SEQ ID No.: 5 of theSequence Listing, it preferably retains about 10%, and preferably about50% or more, of the enzyme activity of a protein having the amino acidsequence described in SEQ ID No.: 5 of the Sequence Listing underconditions of 50° C. and pH 8.

As has been described above, in the case of having isolated a DNAderived from, for example, Corynebacterium glutamicum strain ATCC 13286,the following DNA can be preferably used in the present invention:

(i) A DNA composed of the base sequence of bases nos. 57 to 1295described in SEQ ID No.: 4 of the Sequence Listing;

(ii) A DNA that hybridizes under stringent conditions with apolynucleotide that consists of a base sequence complementary to thebase sequence of bases nos. 57 to 1295 described in SEQ ID No.: 4 of theSequence Listing, and encodes protein having an L-amino acid amidehydrolase activity that catalyzes a reaction that produces a dipeptidefrom an L-amino acid amide and an L-amino acid;

(iii) A DNA that encodes a protein having the amino acid sequencedescribed in SEQ ID No.: 5 of the Sequence Listing; and,

(iv) A DNA that encodes a protein having an amino acid sequence thatcontains a substitution, deletion, insertion, addition or inversion ofone or a plurality of amino acids in the amino acid sequence describedin SEQ ID No.: 5 of the Sequence Listing, and having an L-amino acidamide hydrolase activity that catalyzes a reaction that produces adipeptide from an L-amino acid amide and an L-amino acid.

[III-2] Production of Transformant

Next, production of a transformant that expresses a protein having anL-amino acid amide hydrolase activity will be explained. Numerousexamples are known of producing enzymes, physiologically activesubstances and other useful proteins by using recombinant DNAtechnology, and the use of recombinant DNA technology allows massproduction of useful proteins that naturally occur only in traceamounts.

Preferable examples of a transformant that can be used in the method ofthe present invention include the transformants capable of expressingthe protein (A), (B) or (C) below:

(A) A protein having the amino acid sequence described in SEQ ID No.: 5of the Sequence Listing,

(B) A protein having an amino acid sequence that contains asubstitution, deletion, insertion, addition or inversion of one or aplurality of amino acids in the amino acid sequence described in SEQ IDNo.: 5 of the Sequence Listing, and having an L-amino acid amidehydrolase activity that catalyzes a reaction that produces a dipeptidefrom an L-amino acid amide and an L-amino acid,

(C) A protein encoded by a DNA that hybridizes under stringentconditions with a polynucleotide composed of a base sequencecomplementary to the base sequence of SEQ ID No.: 4 of the SequenceListing, and encodes a protein having an L-amino acid amide hydrolaseactivity that catalyzes a reaction that produces a dipeptide from anL-amino acid amide and an L-amino acid.

To produce a transformant that expresses a protein of the aforementioned(A) to (C) having an L-amino acid amide hydrolase activity, it sufficesthat the DNA of (i) to (iv) indicated in the aforementioned section[III-1] should be inserted into host cells. Namely, the DNA of (i),(ii), (iii) or (iv) is incorporated into an expression vector capable ofexpressing in the host cells followed by introduction of the expressionvector into host cells.

In the case of mass-production of a protein using recombinant DNAtechnology, conjugating the protein within a transformant that producesthe protein to form an inclusion body of protein is also a preferablemode for carrying out the present invention. Advantages of thisexpression and production method include protection of the targetprotein from digestion by proteases present within microbial cells, andsimple purification of the target protein by crushing of the microbialcells followed by centrifugal separation.

The protein inclusion bodies obtained in this manner are converted to aproperly folded, physiologically active protein after going through anactivity regeneration procedure consisting primarily of solubilizing theprotein with a protein denaturant followed by removal of the denaturant.There are numerous examples of this, including regeneration of theactivity of human interleukin-2 (Japanese Patent Application Laid-openPublication No. S61-257931).

In order to obtain an active protein from the inclusion bodies ofprotein, a series of operations including solubilization and activityregeneration are required, and the procedure is more complex than in thecase of producing the active protein directly. However, in the case ofmass-producing a protein that has a detrimental effect on microbialgrowth within microbial cells, that effect can be suppressed byaccumulating the protein in the form of inactive inclusion bodies ofprotein within the microbial cells.

Examples of methods for mass-producing a target protein in the form ofinclusion bodies include a method in which a target protein is expressedindependently under the control of a powerful promoter, and a method inwhich a target protein is expressed in the form of a fused protein fusedwith a protein that is known to be expressed in large amounts.

Moreover, it is also effective to arrange the recognition sequence of arestricting protease at a suitable location in order to cut out thetarget protein following expression in the form of a fused protein.

In the case of mass-production of a protein using recombinant DNAtechnology, although examples of host cells that are transformed includebacterial cells, Actinomyces cells, yeast cells, mold cells, plant cellsand animal cells, intestinal bacteria such as Escherichia coli arecommonly used, with Escherichia coli being used preferably. This isbecause there are numerous pieces of information that are availableregarding techniques for mass-production of a protein using Escherichiacoli. The following provides an explanation of one mode of a method forproducing an L-amino acid amide hydrolase using transformed Escherichiacoli.

Promoters normally used in heterogeneous protein production inEscherichia coli can be used as a promoter for expressing a DNA encodingan L-amino acid amide hydrolase. Examples of the promoters includepowerful promoters such as T7 promoter, trp promoter, lac promoter, trcpromoter, tac promoter, lambda phage P_(R) promoter and P_(L) promoter.

To produce an L-amino acid amide hydrolase in the form of an inclusionbody of fused protein, a gene that encodes another protein, andpreferably a hydrophilic peptide, may be ligated upstream or downstreamof the L-amino acid amide hydrolase gene to obtain a fused protein gene.Such a gene that encodes another protein may be one that provides anincreased accumulation amount of the fused protein and an enhancedsolubility of the fused protein after the denaturation and regenerationsteps. Candidates for such a gene include, for example, T7 gene 10,β-galactosidase gene, dehydrofolate reductase gene, interferon γ gene,interleukin-2 gene and prochymosin gene.

When ligating these genes to a gene that encodes an L-amino acid amidehydrolase, the codon reading frames are made to match. They may eitherbe ligated at a suitable restrictase site or a synthetic DNA of asuitable sequence may be used.

In addition, in order to increase the amount of production, it may bepreferable to ligate a transcription terminating sequence in the form ofa terminator downstream of the fused protein gene. Examples of thisterminator include T7 terminator, fd phage terminator, T4 terminator,tetracycline resistance gene terminator and Escherichia coli trpA geneterminator.

So-called multi-copy vectors are preferable as the vector forintroducing into Escherichia coli a gene that encodes an L-amino acidamide hydrolase or a fused protein consisting of an L-amino acid amidehydrolase and another protein, examples of which include plasmids havinga replication starting point originating in ColE1 such as pUC plasmid,pBR322 plasmid or derivatives thereof. A “derivative” refers to theresult of modifying the plasmid by base substitution, deletion,insertion, addition or inversion. Furthermore, the modification referredto here includes modification resulting from mutagenesis treatment by amutagen or UV irradiation, or spontaneous mutation. More specifically,examples of vectors that can be used include pUC19, pUC18, pBR322,pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219 andpMW218. Further, vectors of phage DNA can also be used.

In addition, the vector preferably has a marker such as an ampicillinresistant gene in order to screen out the transformant. Expressionvectors having powerful promoters are commercially available for use assuch plasmids (such as pUC vector (manufactured by Takara Shuzo), pPROKvector (manufactured by Clontech) and pKK233-2 vector (manufactured byClontech).

A recombinant DNA is obtained by ligating a DNA fragment to a vectorDNA. In this case, a promoter, a gene encoding L-amino acid amidehydrolase or a fused protein consisting of an L-amino acid amidehydrolase and another protein, and depending on the case, a terminatorare ligated in that order.

Transformation of Escherichia coli using the recombinant DNA andculturing the resulting Escherichia coli results in expression andproduction of an L-amino acid amide hydrolase or a fused proteinconsisting of an L-amino acid amide hydrolase and another protein.Although a strain that is normally used in the expression of aheterogeneous gene can be used as a host to be transformed, Escherichiacoli strain JM109, for example, is preferable. Methods for carrying outtransformation and methods for screening out transformants are describedin Molecular Cloning, 2nd Edition, Cold Spring Harbor Press (1989) andother publications.

When expressed as a fused protein, the L-amino acid amide hydrolase maybe cut out with a restriction protease that uses a sequence not presentin the L-amino acid amide hydrolase, such as blood coagulation factor Xaor kallikrein, as a recognition sequence.

A medium normally used for culturing Escherichia coli, such asM9-casamino acid medium or LB medium, may be used for the productionmedium. In addition, culturing conditions and production inductionconditions are suitably selected depending on the marker of the vectorused, promoter, type of host microbe and so forth.

The following method can be used to recover the L-amino acid amidehydrolase or fused protein consisting of an L-amino acid amide hydrolaseand another protein. If the L-amino acid amide hydrolase or its fusedprotein has been solubilized within the microbial cells, after therecovery of the microbial cells, the microbial cells are crushed orlysed so that they can be used as a crude enzyme liquid. Moreover, theL-amino acid amide hydrolase or its fused protein can be purified priorto use by ordinary techniques such as precipitation or filtration,column chromatography as necessary. In this case, a purification methodcan also be used that uses antibody of the L-amino acid amide hydrolaseor its fused protein.

In the case where inclusion bodies of protein are formed, the inclusionbodies are solubilized with a denaturant. Although they may besolubilized with the microbial cell protein, in consideration of thefollowing purification procedure, the inclusion bodies are preferablytaken out and then solubilized. Known methods may be used to recover theinclusion bodies from the microbial cells. For example, inclusion bodiescan be recovered by crushing the microbial cells followed by centrifugalseparation. Examples of denaturants capable of solubilizing inclusionbodies of protein include guanidine hydrochloride (for example, 6 molars(M), pH 5 to 8) and urea (for example, 8 M).

Protein having activity is regenerated by removing these denaturants bydialysis, for example. Tris-HCl buffer solution or phosphate buffersolution and so forth may be used as the dialysis solution used indialysis, and the concentration may be, for example, 20 millimolars (mM)to 0.5 M, while the pH may be, for example, 5 to 8.

The protein concentration during the regeneration step is preferablyheld to about 500 μg/ml or less. The dialysis temperature is preferablyequal to or lower than 5° C. to inhibit the occurrence ofself-crosslinking by the regenerated L-amino acid amide hydrolase.Moreover, in addition to dialysis, dilution or ultrafiltration may beused to remove the denaturants, and regeneration of the enzyme activitycan be expected by employing any one of these method.

In the case of using the DNA indicated in SEQ ID No.: 4 of the SequenceListing for the DNA that encodes an L-amino acid amide hydrolase, theL-amino acid amide hydrolase that has the amino acid sequence describedin SEQ ID No.: 5 is produced.

It should be noted that genetic engineering techniques can be carriedout in accordance with the techniques described in the literature suchas Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

[IV] Dipeptide Production Method

The dipeptide production method of the present invention produces adipeptide from an L-amino acid amide and an L-amino acid using an enzymeor enzyme-containing substance having the ability to form a dipeptidefrom an L-amino acid amide and an L-amino acid, and more specifically, aculture of microbes, microbial cells separated from the culture ortreated microbial cells from the microbe.

The aforementioned L-amino acid amide hydrolase has activity that formsan L-amino acid by hydrolyzing L-amino acid amide, and activity thatproduces a dipeptide by using an L-amino acid amide and an L-amino acidas substrates.

FIG. 1 is a flowchart of the dipeptide production method of the presentinvention.

First, microbes having the ability to form a dipeptide from an L-aminoacid amide and an L-amino acid are cultured in a medium, and an L-aminoacid amide hydrolase is produced and accumulated in the culture and/orcells (Step S1).

Next, a purified L-amino acid amide hydrolase is produced by recoveringand purifying the L-amino acid amide hydrolase (Step S2).

Subsequently, a dipeptide can be produced in large amounts by adding theL-amino acid amide and the L-amino acid to the purified L-amino acidamide hydrolase produced in Step S2 or the L-amino acid amide hydrolaseaccumulated in Step S1, and allowing reaction to proceed (Step S3).

For the method by which the L-amino acid amide hydrolase produced by theaforementioned microbes is allowed to act on the L-amino acid amide andthe L-amino acid, the substrates may be added either directly to theculture liquid while culturing the aforementioned microbes, or microbialcells may be separated from the microbial culture by centrifugation andso forth, followed by resuspending them in buffer either directly orafter washing, and then adding L-amino acid amide and L-amino acidfollowed by allowing the resultant to react. Alternatively, microbialcells can be used that have been immobilized by known methods usingpolyacrylamide gel, carrageenan, alginic acid gel and the like.

In addition, crushed microbial cells, acetone-treated microbial cells orfreeze-dried microbial cells may be used as the treated microbial cellproduct. Methods such as ultrasonic crushing, French press crushing andglass bead crushing are used for crushing microbial cells, while methodsusing egg white lysozyme, peptidase treatment or a suitable combinationthereof are used in the case of lysing microbial cells.

Moreover, L-amino acid amide hydrolase may be recovered from the treatedmicrobial cell product and used as a crude enzyme liquid, or the enzymemay be purified before use as necessary. Ordinary enzyme purificationmethods can be used for purifying the enzyme obtained from a culture.More specifically, microbial cells are collected by centrifugalseparation and so forth, the cells are then crushed by mechanicalmethods such as ultrasound treatment, glass beads or a dynomill, andsolid materials such as cell fragments are removed by centrifugalseparation to obtain crude enzyme followed by purification of theaforementioned L-alanine amide hydrolase by performingultracentrifugation fractionation, salting out, organic solventprecipitation, ion exchange chromatography, adsorption chromatography,gel filtration chromatography, hydrophobic chromatography and so forth.

Namely, in the case of a fraction having activity to form a dipeptidefrom an L-amino acid amide and an L-amino acid, the entire enzyme andenzyme-containing substance can be used. Here, an “enzyme-containingsubstance” refers to any substance that contains the enzyme, andincludes specific forms such as a culture of microbes that produce theenzyme, microbial cells separated from the culture and treated microbialcells. A culture of microbes refers to a thing that is obtained byculturing microbes, and more specifically, refers to a mixture ofmicrobial cells, the medium used to culture the microbes and substancesproduced by the cultured microbes. In addition, the microbial cells maybe washed and used as washed microbial cells, or immobilized cells maybe used that have been immobilized by covalent bonding, adsorption orinclusion methods. In addition, since some microbes are partially lysedduring culturing depending on the microbes used, the culture supernatantmay also be used as the enzyme-containing substance in such cases.

The amount of enzyme or enzyme-containing substance used should be anamount at which the target effect is demonstrated (hereinafter,“effective amount”), and although this effective amount can be easilydetermined through simple, preliminary experiment by a person withordinary skill in the art, in the case of using washed cells, forexample, the amount used is 1 to 500 grams (g) per liter of the reactionmixture.

Any L-amino acid amide can be used for the L-amino acid amide so far asit is an L-amino acid amide that can be hydrolyzed at the substratespecificity of the L-amino acid amide hydrolase. Examples of the L-aminoacid amide include not only L-amino acid amides corresponding tonaturally-occurring amino acids, but also L-amino acid amidescorresponding to non-naturally-occurring amino acids or theirderivatives. In addition, since the L-amino acid amide hydrolase used inthe present invention imparts L-amino acid by asymmetrically hydrolyzinga racemic L-amino acid amide, racemic amino acid amides that can beinexpensively synthesized with the Strecker method can also be used. Inthe present invention, preferable examples of L-amino acid amidesinclude L-alanine amide, glycine amide and L-aspartic acid amide, withL-alanine amide being particularly preferable.

There is no particular restriction on the L-amino acid so far as it canform a dipeptide with an L-amino acid amide at a substrate specificityof the L-amino acid amide hydrolase, and known L-amino acids can beused. Preferable examples of L-amino acids include L-glutamine,L-asparagine, glycine, L-alanine, L-valine, L-leucine, L-isoleucine,L-methionine, L-proline, L-phenylalanine, L-tryptophan, L-serine,L-threonine, L-tyrosine, L-lysine, L-arginine, L-histidine andL-glutamine, with L-glutamic acid and L-asparagine being particularlypreferable.

Dipeptides may be produced by selecting one type each of theaforementioned L-amino acid amide and L-amino acid, or dipeptides may beproduced by selecting two or more types.

Concentrations of the L-amino acid amide and L-amino acid used asstarting materials are each 1 mM to 10 M, and preferably 0.1 M to 2 M.However, in some cases, it is preferable to add an amount of L-aminoacid equal to or greater than the amount of L-amino acid amide. Inaddition, when needed, for example, in the case where highconcentrations of substrates inhibit the reaction, these can besuccessively added during the reaction after adjusting them toconcentrations that do not result in inhibition.

The reaction temperature is 10 to 70° C., and preferably 20 to 50° C.,while the reaction pH is 2 to 12, and preferably 3 to 11. By carryingout the reaction in this manner for about 2 to 48 hours, dipeptide isproduced and accumulates in the reaction mixture. Since the dipeptideproduction reaction is an equilibrium reaction, in order to achieveefficient production, the reaction is allowed to proceed further byseparating the dipeptide and ammonia produced.

EXAMPLES

Hereinafter, the present invention will be explained in more detail byway of examples. However, the present invention should not be consideredto be limited to these examples. Note that in the examples, quantitativedetermination of L-alanine, L-alanyl-L-glutamine orL-alanyl-L-asparagine was carried out by a method using high-performanceliquid chromatography (column: Inertsil ODS-2 manufactured by GLScience, eluate: aqueous phosphate solution (pH 2.1), 2.5 mM sodium1-octanesulfonate/methanol=10/1, flow rate: 1.0 mL/min, detection: 210nanometers (nm)).

Example 1 Production of L-Alanyl-L-Asparagine

A 50 milliliter (ml or mL) aliquot of a medium containing 0.5% (w/v)yeast extract, 0.5% (w/v) peptone, 0.5% (w/v) glycerol, 0.5% (w/v)sodium chloride and 0.5% (w/v) L-alanine amide hydrochloride (pH 7.0)was dispensed to a 500 mL Sakaguchi flask and sterilized at 120° C. for20 minutes. One loopful of the microbes shown in Table 1, which werecultured at 30° C. for 24 hours on a slant medium containing 0.5% (w/v)yeast extract, 0.5% (w/v) peptone, 0.5% (w/v) glycerol, 0.5% (w/v)sodium chloride, 0.5% (w/v) L-alanine amide hydrochloride and 2% (w/v)agar (pH 7.0), was inoculated into the aforementioned medium andcultured by shake culturing for 20 hours at 30° C. and 120strokes/minute. Following the culturing, the microbial cells wereseparated by centrifugation, washed twice with an amount ofphysiological saline equal to the amount of culture liquid andcentrifuged again to collect the microbial cells followed by suspendingwith 0.2 M Tris-HCl buffer (pH 9.0) to a final volume of 10 mL. 1 mL ofthis microbial cell suspension was then added to 4 mL of theaforementioned buffer containing 62.5 mM L-alanine amide hydrochlorideand 250 mM L-asparagine, and after bringing to a total volume of 5 mL,was allowed to react at 30° C. for 24 hours. A lot in which microbialcells were not added was established as a control experiment. Theresults are shown in Table 1. TABLE 1 L-Ala-L-Asn Microbe produced (mM)Bacillus megateirum FERM BP-8090 0.4 Corynebacterium glutamicumATCC13286 1.8 Erwinia carotovora FERM BP-8089 0.5 Rhodococcusrhodochrous ATTC19149 1.0 Chryseobacterium meningosepticum ATTC13253 0.1Micrococcus luteus ATCC9341 0.1 Pseudomonas saccharophila ATCC9114 0.1Cryptococcus albidus IFO610 1.8 Trichosporon gracile ATCC24660 2.5Rhodosporidium diobovatum ATCC22264 2.7 Sporobolomyces salmonicolorIFO1038 1.5 Tremela foliacea IFO9297 3.3 Torulaspora delbrueckii IFO10832.9 Sterigmatomyces elviae IFO1843 0.1 Rhodotorula ingeniosa ATCC229930.1 Microbial cells not added Below detection limitL-Ala-L-Asn: L-alanyl-asparagine

Example 2 Purification of L-Alanine Amide Hydrolase from Corynebacteriumglutamicum Strain ATCC 13286

Measurement of enzyme titer was carried out in the manner describedbelow. 200 micromoles (μmol) of Tris-HCl buffer (pH 9.0), 50 μmol ofL-alanine amide hydrochloride and a suitable amount of enzyme liquidwere added and mixed to bring to a final volume of 1 ml, and allowed toreact at 30° C. for 60 minutes. Then, 4 ml of aqueous phosphate solution(pH 2.1) was added to stop the reaction. The L-alanine produced wasquantified by high-performance liquid chromatography. The amount ofenzyme that produced 1 μmol of L-alanine in 1 minute was defined as 1unit of enzyme.

8 liters (L) of Corynebacterium glutamicum strain ATCC 13286 wascultured in the same manner as Example 1 followed by collection of themicrobial cells by centrifugal separation. The following procedure wascarried out on ice or at 4° C. After washing the microbial cells with 50mM potassium phosphate buffer (pH 7.0), the cells were subjected tocrushing treatment for about 10 minutes using glass beads having adiameter of 0.1 mm. The glass beads and crushed cell liquid were thenseparated, and the crushed cell fragments were removed by centrifugalseparation for 30 minutes at 20,000×g to obtain a cell-free extract.Moreover, the insoluble fraction was removed by ultracentrifugation for60 minutes at 200,000×g to obtain a soluble fraction in the form of thesupernatant. Ammonium sulfate was then added to the resulting solublefraction to 60% saturation followed by recovery of the precipitate bycentrifuging for 30 minutes at 20,000×g. The resulting precipitate wasdissolved in a small amount of 50 mM potassium phosphate buffer (pH 7.0)and then dialyzed against 50 mM potassium phosphate buffer (pH 7.0).This enzyme liquid was then applied to a Q-Sepharose HP columnpre-equilibrated with 50 mM potassium phosphate buffer (pH 7.0), and theenzyme was eluted over a linear concentration gradient of 50 mMpotassium phosphate buffer (pH 7.0) containing 0 to 1.0 M sodiumchloride. The active fraction was collected and applied to a Superdex200pg column pre-equilibrated with 50 mM potassium phosphate buffer (pH7.0), and the enzyme was then eluted with the same buffer. The activefraction was collected and dialyzed against 20 mM potassium phosphatebuffer (pH 7.0) containing 0.5 M ammonium sulfate, and then applied to aPhenyl-Sepharose HP column pre-equilibrated with 20 mM potassiumphosphate buffer (pH 7.0) containing 0.5 M ammonium sulfate. The enzymewas then eluted over a linear concentration gradient of 20 mM potassiumphosphate buffer (pH 7.0) containing 0.5 to 0 M ammonium sulfate. Theactive fraction was collected and dialyzed against 50 mM potassiumphosphate buffer (pH 7.0), and this was then applied to a MonoQ columnpre-equilibrated with 50 mM potassium phosphate buffer (pH 7.0), enzymewas eluted over a linear concentration gradient of 50 mM potassiumphosphate buffer (pH 7.0) containing 0 to 1.0 M sodium chloride.L-alanine amide hydrolase was uniformly purified on the basis ofelectrophoresis in this manner. The total amounts of protein andspecific activities in each purification step are shown in Table 2.TABLE 2 Total Total Specific activity protein activity Step (unit) (mg)(unit/mg) Cell-free extract 80 2000 0.040 Soluble fraction 71 1690 0.042Ammonium Sulfate fraction 79 1080 0.073 Q-Sepharose HP 56 379 0.149Superdex200pg 21 151 0.135 Phenyl-Sepharose HP 12.5 6.60 1.897 MonoQ 2.40.24 9.841

Example 3 Evaluation of Molecular Weight of L-Alanine Hydrolase

The equivalent of 0.5 microgram (μg) of the purified enzyme preparationobtained according to the method of Example 2 was applied topolyacrylamide electrophoresis. 0.3% (w/v) Tris, 1.44% (w/v) glycine and0.1% (w/v) sodium lauryl sulfate were used for the electrophoresisbuffer, concentration gradient gel having a gel concentration of 10 to20% (Multigel 10 to 20, manufactured by Daiichi Pure Chemicals) was usedfor the polyacrylamide gel, and precision pre-stained standards(manufactured by Biorad) were used for the molecular weight markers.Following completion of electrophoresis, the gel was stained withCoomassie brilliant blue R-250, and a uniform band was detected at thelocation calculated to have a molecular weight of 42,000 to 46,000.

Example 4 Evaluation of Optimum pH of L-Alanine Amide Hydrolase

L-alanine amide was hydrolyzed using the L-alanine amide hydrolaseuniformly purified in Example 2 followed by evaluation of the pH for thereaction that produces L-alanine in the manner described below. 200 μmolof buffer solution consisting of sodium acetate buffer (pH 3.0 to 6.0),potassium phosphate buffer (pH 6.0 to 8.0), Tris-HCl buffer (pH 7.0 to9.0), sodium carbonate buffer (pH 8.0 to 10.0) or glycine-sodiumhydroxide buffer, 50 μmol of L-alanine amide hydrochloride and asuitable amount of enzyme liquid were added and mixed to a final volumeof 1 ml followed by allowing to react at 30° C. for 60 minutes andevaluating enzyme activity. Those results when assigning a value of 100% to activity in the case of using Tris-HCl buffer (pH 8.0) are shownin FIG. 2.

Example 5 Evaluation of L-Alanine Amide Hydrolase Reaction Temperature

L-alanine amide was hydrolyzed using the L-alanine amide hydrolaseuniformly purified in Example 2, followed by evaluation of the reactiontemperature for the reaction that produces L-alanine in the mannerdescribed below. 200 μmol of Tris-HCl buffer, 50 μmol of L-alanine amidehydrochloride and a suitable amount of enzyme were added and mixed to afinal volume of 1 ml, and then allowed to react at a temperature of 25,30, 40, 50 or 60° C. for 60 minutes followed by evaluation of enzymeactivity. Those results when assigning a value of 100% to activity inthe case of a reaction temperature of 40° C. are shown in FIG. 3.

Example 6 Production of L-Alanyl-L-Asparagine and L-Alanyl-L-Glutamine

The L-alanine amide hydrolase uniformly purified in Example 2 wasallowed to act on L-alanine amide hydrochloride and L-asparagine orL-alanine amide hydrochloride and L-glutamine to produceL-alanyl-L-asparagine or L-alanyl-L-glutamine. In the case of obtainingL-alanyl-L-asparagine, 200 μmol of Tris-HCl buffer (pH 9.0), 50 μmol orL-alanine amide hydrochloride, 150 μmol of L-asparagine and enzymeliquid containing 0.08 unit of L-alanine amide hydrolase were added andmixed to a final volume of 1 ml. In the case of obtainingL-alanyl-L-glutamine, the reactants were mixed under the same conditionsas in the case of L-alanyl-L-asparagine with the exception of using 150μmol of L-glutamine instead of 150 μmol of L-asparagine. Controlexperiments were established for the case of using one of the substratesor setting the lot in which enzyme was not added. The reaction wascarried out at a reaction temperature of 30° C. for 10 hours, and theresults of quantifying the target product are shown in Table 3. TABLE 3Product Enzyme concentration Substrate addition Product (mM) L-alanineamide L-asparagine Yes L-alanyl- 8.4 L-asparagine L-alanine amide — YesL-alanyl- Below L-asparagine detection limit — L-asparagine YesL-alanyl- Below L-asparagine detection limit L-alanine amideL-asparagine No L-alanyl- Below L-asparagine detection limit L-alanineamide L-glutamine Yes L-alanyl- 7.7 L-glutamine L-alanine amide — YesL-alanyl- Below L-glutamine detection limit — L-glutamine Yes L-alanyl-Below L-glutamine detection limit L-alanine amide L-glutamine NoL-alanyl- Below L-glutamine detection limit

Example 7 Isolation of L-Alanine Amide Hydrolase Gene

Hereinafter, isolation of L-alanine amide hydrolase gene and expressionof L-alanine amide hydrolase in Escherichia coli (E. coli), will bedescribed. Corynebacterium glutamicum strain ATCC 13286 was used for themicrobe strain. E. coli JM109 was used for the host and pUC18 was usedfor the vector for both gene isolation and expression of L-alanine amidehydrolase.

1. Production of PCR Primer Based on Determined Amino Acid Sequence

Mixed primers having the base sequence indicated in SEQ ID No.: 2 andSEQ ID No.: 3, respectively, were produced based on N-terminal aminoacid sequence of L-alanine amide hydrolase originating in theaforementioned Corynebacterium glutamicum strain ATCC 13286.

2. Acquisition of Microbial cells

Corynebacterium glutamicum strain ATCC 13286 was cultured on CM2Gly agarmedium (0.5 g/dl glycerol, 1.0 g/dl yeast extract, 1.0 g/dl peptone, 0.5g/dl NaCl, 2 g/dl agar, pH 7.0) at 30° C. for 24 hours to refresh themicrobe. One loopful thereof was then inoculated in a 500 ml-volumeSakaguchi flask containing 50 ml of CM2Gly liquid medium, followed byshake culturing at 30° C. for 16 hours under aerobic conditions.

3. Acquisition of Chromosomal DNA from Microbial Cells

50 ml of culture liquid were centrifuged (12,000 rounds per minute(rpm), 4° C., 15 minutes) to collect the microbial cells. Thesemicrobial cells were then suspended in 10 ml of 50 mM Tris-HCl buffer(pH 8.0) containing 20 mM EDTA followed by recovery of the microbialcells by centrifugal separation. The microbial cells were againsuspended in 10 ml of 50 mM Tris-HCl buffer (pH 8.0) containing 20 mMEDTA. Moreover, after adding 0.5 ml of 20 mg/ml lysozyme solution and 1ml of 10% SDS (sodium dodecyl sulfate) solution to this suspension, thesolution was incubated at 55° C. for 20 minutes. The incubated solutionwas then deproteinized by the addition of an equal volume of phenolsaturated with 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA. Anequal volume of 2-propanol was added to the separated aqueous layer toprecipitate a DNA followed by recovery of that precipitated DNA. Afterdissolving the DNA precipitated in 0.5 ml of 50 mM Tris-HCl buffer (pH8.0) containing 20 mM EDTA, 5 microliters (II) of 10 mg/ml RNase and 5μl of 10 mg/ml Proteinase K were added and allowed to react at 55° C.for 2 hours. After the reaction, this solution was deproteinized by theaddition of an equal volume of phenol saturated with 10 mM Tris-HClbuffer (pH 8.0) containing 1 mM EDTA. Moreover, an equal volume of 24:1chloroform/isoamyl alcohol was added to the separated aqueous layer torecover the aqueous layer. To the aqueous layer obtained by doing theprocedure two times, 3 M sodium acetate solution (pH 5.2) was added tobring to a final concentration of 0.4 M and 2 volumes of ethanol wasadded. The DNA that was formed as a precipitate was recovered, and afterwashing with 70% ethanol, was dried and dissolved in 1 ml of 10 mMTris-HCl buffer (pH 8.0) containing 1 mM EDTA.

4. Acquisition of DNA Fragment Containing a Portion of L-Alanine AmideHydrolase Gene by Cassette PCR Method

The TaKaRa LA PCR In Vitro Cloning Kit (Takara Shuzo) was used forisolation and amplification of DNA molecules containing a gene (aah)encoding L-alanine amide hydrolase using the cassette PCR method. Unlessotherwise indicated specifically, the experiment was carried out basedon the method described in the manual. In the cassette PCR method, incase of using Primer 1 (1st PCR, SEQ ID No.: 2) and Primer 2 (2nd PCR,SEQ ID No.: 3) as primers, a roughly 0.5 kilobase (kb) band (Fragment 1)was amplified with the Eco RI cassette. As a result of determining thebase sequence of this fragment, Fragment 1 was verified to be a portionof aah.

5. Cloning of L-Alanine Amide Hydrolase Gene from Gene Library

In order to acquire the entire length of aah, Southern hybridization wascarried out first using Fragment 1 as a probe.

The DNA fragment to serve as the probe was prepared to about 50nanogram/microliters (ng/μl) and the probe was labeled by incubating 16μl of this DNA solution at 37° C. for 24 hours in accordance with theprotocol using DIG High Prime (Boehringer-Mannheim).

1 μg of chromosomal DNA was completely digested by combining variousrestrictases, electophoresed with 0.8% agarose gel, and then blottedonto Nylon membranes (Boehringer-Mannheim, positively charged Nylonmembranes). Southern hybridization was then carried out in accordancewith the established method. Hybridization was carried out using DIGEasy Hyb (Boehringer-Mannheim), and after hybridizing at 50° C. for 30minutes, the probe was added following by hybridizing at 50° C. for 18hours. Detection was carried out using the DIG Nucleotide Detection Kit(Boehringer-Mannheim).

As a result, a band was detected at about the 7 kb position in the BglII severed product. This 7 kb domain fragment was collected and coupledto pUC18 to produce a library (120 strains) with E. coli JM109. Colonyhybridization was then carried out in accordance with the establishedmethods. The colonies were then transferred to Nylon membrane filters(Boehringer-Mannheim, Nylon membranes for colony and plaquehybridization) followed by alkali denaturation, neutralization andimmobilization treatment. Hybridization was carried out using DIG EasyHyb. The filter was immersed in a buffer and pre-hybridized at 42° C.for 30 minutes. Subsequently, the aforementioned labeled probe wasadded, followed by hybridization at 42° C. for 18 hours. After washingwith SSC buffer, one positive clone was selected using the DIGNucleotide Detection Kit.

6. Base Sequence of L-Alanine Amide Hydrolase Gene Derived fromCorynebacterium glutamicum Strain ATCC 13286

Plasmids retained by the selected transformant were prepared inaccordance with the method described in Molecular Cloning, 2nd edition,Cold Spring Harbor Press (1989), and the base sequence in the vicinitythat hybridized with the probe was determined. An open reading frame(ORF) was present that encoded protein containing 30 residues of theN-terminal amino acid sequence of L-alanine amide hydrolase, and wasverified to be the gene aah that encodes L-alanine amide hydrolase. Thebase sequence of the full-length L-alanine amide hydrolase gene is shownin SEQ ID No.: 4 of the Sequence Listing. When the homology of theresulting ORF was examined using Genetyx, the base sequence exhibited ahomology of 57.6% with the known proline iminopeptidase derived fromPropionibacterium

bacteria.

Example 8 Expression of L-Alanine Amide Hydrolase Gene in E. coli

Plasmid pUCAAH coupled to aah downstream of the lac promoter of pUC18was constructed in order to express aah in E. coli. Fragments amplifiedby PCR using the chromosomal DNA of Corynebacterium glutamicum strainATCC 13286 as template and the oligonucleotides shown in Table 4 asprimers were treated with Sac I and Sma I, and after ligating to the SacI- and Sma I-cleaved product of pUC18, were used to transform E. coliJM109. Strains having the target plasmid were selected fromampicillin-resistant strains, and the constructed expression plasmid wasdesignated as pUCAAH. TABLE 4 Primers Used to Construct L-Alanine AmideHydrolase Expression Vector Primer Sequence 5′ sideGGCGAGCTCGGGCAGTGGTGGGGGTGGTGT    Sac I (SEQ ID No.: 6) 3′ sideCGGGGGCCCTCAGCGTACCTCTCGGCCGTG    Sma I (SEQ ID No.: 7)

The transformant expressing L-alanine amide hydrolase in E. coli havingpUCAAH was seed cultured at 37° C. for 16 hours in LB medium containing0.1 mg/ml ampicillin. 1 ml of this pre-culture liquid was seeded into a500 ml Sakaguchi flask containing 50 ml of LB medium followed by finalculturing at 37° C. Two hours after the start of culturing,isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to a finalconcentration of 1 mM followed by additionally culturing for 3 hours.Following completion of the culturing, the microbes were collected andwashed, suspended in 10 ml of 20 mM phosphate buffer (pH 8.0), and thensubjected to ultrasonic crushing for 30 minutes at 180 W. The solutionwas recovered and centrifuged for 10 minutes at 12,000 rpm, and theresulting supernatant was used as a cell-free extract.

Example 9 Measurement of Activity of L-Alanine Amide Hydrolase

After completion of the culturing, a cell-free extract was prepared andthe activity of L-alanine amide hydrolase was measured using this as theenzyme source. Measurement of L-alanine amide enzyme activity wascarried out by incubating a reaction mixture containing 50 mM L-alanineamide, 150 mM L-glutamine, 100 mM Tris-HCl buffer (pH 9.0), 10 mM EDTAand enzyme solution at 30° C. for 60 minutes followed by stopping thereaction by adding aqueous phosphoric acid (pH 1.5) equal to fourvolumes of the reaction mixture. The amount of L-alanyl-L-glutamine wasdetermined by HPLC. For the unit of enzyme activity, enzyme activitythat produces 1 μmol of L-alanyl-L-glutamine in 1 minute under theseconditions was defined as 1 unit (U).

The conditions of HPLC used for analysis were as indicated below.

Column: Inertsil ODS-2

Mobile phase: (aqueous phosphoric acid solution (pH 2.1)), 2.5 mMsodium-1-octanesulfonate/methanol=10/1

Column temperature: 40° C.

Flow rate: 1.0 ml/minute

Detection: UV, 210 nm

As a result, 0.05 U/mg of L-alanine amide hydrolase activity wasdetected in the case of inserting pUC18 AAH, thereby confirming that thecloned aah gene was expressed in E. coli. Furthermore, no activity wasdetected when only pUC18 was inserted as a control.

Example 10 Expression of His-Tag L-Alanine Amide Hydrolase Gene in E.coli

Plasmid pQEAAH that expresses L-alanine amide hydrolase as an His-Taqprotein downstream of the lac promoter of pUC18 was constructed toexpress aah in E. coli. Fragments amplified by PCR using the chromosomalDNA of Corynebacterium glutamicum strain ATCC 13286 as template and theoligonucleotides shown in Table 5 as primers were treated with Sac I andSma I, and ligated to the Sac I- and Sma I-cleaved products of pQE-30(Qiagen), and the resultant was used to transform E. coli JM109. Strainshaving the target plasmid were selected from ampicillin-resistantstrains, and the constructed expression plasmid was designated asPQEAAH. TABLE 5 Primers Used to Construct His-Tag L-Alanine AmideHydrolase Expression Vector Primer Sequence 5′ side GGC GAG CTC ATG ACTAAA ACA CTT GGT TCC     Sac I (SEQ ID No.: 8) 3′ side CGG GGG CCC TCAGCG TAC CTC TCG GCC GTG       Sma I (SEQ ID No.: 7)

When the activity of the transformant expressing L-alanine amidehydrolase in E. coli having pQEAAH was measured in the same manner aspreviously described, it was found to exhibit L-alanine amide hydrolaseactivity of 0.48 U/mg.

Example 11 Preparation of His-Tag Purified Enzyme

Microbial cells from 150 ml of culture broth of E. coli JM109 havingpQEAAH were crushed according to the aforementioned method, and anHis-Tag L-alanine amide hydrolase was purified using the His Trap Kit(manufactured by Amersham Pharmacia Biotech) according to the protocolprovided with the kit. 24 milligrams (mg) of protein was acquired thatexhibited a single band on SDS-PAGE, and the specific activity of theL-alanine amide hydrolase of that protein was 13.4 U/mg. The productionyield of Ala-Gln was 7.2% relative to L-alanine amide.

Example 12 Study of Substrate Specificity Using His-Tag Purified Enzyme

The synthesis of peptides other than the L-alanyl-L-asparagine andL-alanyl-L-glutamine indicated in Example 6 by the acquired L-alanineamide hydrolase was studied using His-Tag purified enzyme.

(1) Peptide Synthesis of L-Alanine Amide and Other L-Amino Acids

The synthesis reaction was carried out by incubating a reaction mixturecontaining 100 mM L-alanine amide, 150 mM test amino acid, 100 mMTris-HCl buffer (pH 9.0), 10 mM EDTA and enzyme solution (0.0045 U/ml)at 25° C. for 3 hours followed by quantification of the peptidesproduced by HPLC. As a result, numerous other peptides were produced asindicated below in addition to L-alanyl-L-asparagine andL-alanyl-L-glutamine. 7.54 mM L-alanyl-glycine was synthesized in thecase of using glycine for the test amino acid, 10.11 mML-alanyl-L-alanine was synthesized in the case of using L-alanine, 9.72mM L-alanyl-L-valine was synthesized in the case of using L-valine, 9.60mM L-alanyl-L-leucine was synthesized in the case of using L-leucine,14.11 mM L-alanyl-L-isoleucine was synthesized in the case of usingL-isoleucine, 14.49 mM L-alanyl-L-methionine was synthesized in the caseof using L-methionine, 0.81 mM L-alanyl-L-proline was synthesized in thecase of using L-proline, 13.42 mM L-alanyl-L-phenylalanine wassynthesized in the case of using L-phenylalanine, 10.09 mML-alanyl-L-tryptophan was synthesized in the case of using L-tryptophan,24.67 mM L-alanyl-L-serine was synthesized in the case of usingL-serine, 20.76 mM L-alanyl-L-threonine was synthesized in the case ofusing L-threonine, 1.52 mM L-alanyl-L-tyrosine was synthesized in thecase of using L-tyrosine, 18.83 mM L-alanyl-L-lysine was synthesized inthe case of using L-lysine, 27.69 mM L-alanyl-L-arginine was synthesizedin the case of using L-arginine, 12.52 mM L-alanyl-L-histidine wassynthesized in the case of using L-histidine, and 1.20 mML-alanyl-L-glutamate was synthesized in the case of using L-glutamicacid.

(2) Peptide Synthesis from Other L-Amino Acids and L-Glutamine

Peptide synthesis was carried out using glycine amide andL-aspartate-α-amide instead of L-alanine amide.

The synthesis reaction was carried out by incubating a reaction mixturecontaining 100 mM test amino acid amide, 150 mM L-glutamine, 100 mMTris-HCl buffer (pH 9.0), 10 mM EDTA and enzyme (0.0045 U/ml) at 25° C.for 3 hours, followed by quantification of the peptides produced byHPLC. As a result, 17.7 mM glycyl-L-glutamine was produced in the caseof using glycine amide. In addition, 21.2 mM α-L-aspartyl-glutamine wasproduced in the case of using L-aspartate-α-amide.

As has been described above, it was clearly demonstrated that theL-alanine amide hydrolase obtained in the manner previously describedwas able to use various types of L-amino acid amides and L-amino acidsas substrates. Consequently, it was clearly determined that theresulting enzyme is more appropriately referred to as an L-amino acidamide hydrolase rather than an L-alanine amide hydrolase.

[Sequence Listing]

-   SEQ ID No.: 1: N-terminal amino acid sequence of L-alanine amide    hydrolase derived from Corynebacterium glutamicum-   SEQ ID No.: 2: PCR primer-   SEQ ID No.:3: PCR primer-   SEQ ID No.: 4: CDS sequence of L-amino acid amide hydrolase derived    from Corynebacterium glutamicum-   SEQ ID No.: 5: Amino acid sequence of L-amino acid amide hydrolase    derived from Corynebacterium glutamicum-   SEQ ID No.: 6: Primer-   SEQ ID No.: 7: Primer-   SEQ ID No.: 8: Primer

INDUSTRIAL APPLICABILITY

According to the dipeptide production method of the present invention, adipeptide can be produced using comparatively inexpensively availableL-amino acid amide and L-amino acid without going through a complexsynthesis method. This makes it possible to reduce the production costof dipeptides useful as pharmaceutical materials, functional foods andso forth. In addition, according to the dipeptide production method ofthe present invention, various types of dipeptides can be produced usingvarious types of L-amino acid amides and L-amino acids as raw materials.In addition, the L-amino acid amide hydrolase of the present inventioncan be advantageously used in the dipeptide production method of thepresent invention.

1-8. (canceled)
 9. An L-amino acid amide hydrolase obtained from a microbe belonging to the genus Erwinia, genus Rhodococcus, genus Chryseobacterium, genus Micrococcus, genus Cryptococcus, genus Trichosporon, genus Rhodosporidium, genus Sporobolomyces, genus Tremela, genus Torulaspora, genus Sterigmatomyces or genus Rhodotorula, which catalyzes a reaction that produces a dipeptide from an L-amino acid amide and an L-amino acid.
 10. A production method of L-amino acid amide hydrolase, comprising: culturing a microbe belonging to the genus Erwinia, genus Rhodococcus, genus Chryseobacterium, genus Micrococcus, genus Cryptococcus, genus Trichosporon, genus Rhodosporidium, genus Sporobolomyces, genus Tremela, genus Torulaspora, genus Sterigmatomyces or genus Rhodotorula in a medium, and accumulating in the medium and/or cells an L-amino acid amide hydrolase that catalyzes a reaction that produces a dipeptide from an L-amino acid amide and an L-amino acid.
 11. (canceled) 